Production and Characterization of Astaxanthin Nanoparticles

There is increasing interest on behalf of consumers and the food industry for the enrichment of common food with health-promoting bioactive molecules. Clinical studies have demonstrated that tangible health benefits may derive from the intake of bioactive compounds, in the prevention of dietary related pathologies such as diabetes, cancer, obesity and cardiovascular diseases. The beneficial effect is usually given by the presence in food of peculiar molecules such us carotenoids, polyphenols, polyunsaturated fatty acids and bioactive peptides, to cite a few. Unfortunately, these compounds display high susceptibility to environmental conditions such as light, extreme pH and temperature, and to standard food manufacturing processes. They can also account for undesirable flavors, colors or affect final product stability and appearance, thus rendering their presence in the product an issue rather than a useful addition. The addition of nutrients in small quantities into a food system may not significantly affect its proprieties, but the high amounts, often required to meet certain health claims and benefits, might bring to a product with a poorly acceptable sensory profile and a scarce stability. In particular, lipophilic bioactive ingredients display a major challenge. Besides their limited solubility in most of the foods and beverages, they are characterized by high susceptibility to oxidation and by a lower adsorption through the gastrointestinal tract in comparison to more hydrophilic substances, meaning a scanty bioavailability. Hence, there is a pressing need for the production of edible delivery systems or carriers that could efficiently encapsulate, protect and improve the handling of lipophilic molecules. The objective of this thesis was to develop a system suitable for the encapsulation of lipophilic molecules, capable of: a) protecting the ingredient from the surrounding environment (extreme pH, heat, UV light, oxygen); b) preserving its functionality (e.g. antioxidant activity); c) reducing the impact on the organoleptic level; d) improving the bioavailability of the encapsulated molecules. This last point in particular could be achieved by using sub-cellular delivery systems referred to as nanoparticles or nanocarriers, which may potentially enhance the transport across the intestinal wall. To this purpose, astaxanthin was chosen as a model bioactive compound. Astaxanthin is a keto-carotenoid that displays several biological activities, such as high antioxidant capacity, that may contribute to the prevention of degenerative pathologies like diabetes, cancer, cardiovascular diseases and chronic bowel disease. However, like all carotenoids it is characterized by a strong lipophilic character that makes its inclusion in many types of aqueous-based foods and beverages rather a challenge. This aspect is the main cause of its poor absorption through the human intestinal mucosae. Moreover, astaxanthin is labile to common processing conditions such as the presence of light and oxygen, extreme pH and high temperatures. For these reasons a lot of efforts have been put in these past years to find suitable solutions for its protection and manipulation. In order to develop the suitable encapsulate, in chapter 2 an emulsification and solvent-evaporation technique was used as encapsulation approach; whey proteins were selected as the matrix to envelope the core constituted by an astaxanthin-enriched oleoresin derived from Haematococcus pluvialis, a microalgae representing the main natural source of the carotenoid. The process was optimized by varying crucial parameters and the stability of the nanoparticles was tested in different conditions. This analysis highlighted a better performance of the encapsulated molecule in comparison to the starting oleoresin. Good release properties during in-vitro simulated digestion and the increase of the solubility in water were observed. In chapter 3, the study was focused on the research for plant alternative proteins as encapsulating matrices in order to satisfy the increasing interest of the consumers for substitutes of animal-deriving ingredients. This allowed to identify pea protein isolate as a valid candidate for the development of a vegetarian/vegan-allergen free nanocarrier. Finally, Chapter 4 dealt in depth with the antioxidant properties displayed by the astaxanthin nanoparticles through in vitro colorimetric assay and by the development of a cell-based assay. The encapsulates showed higher antioxidant capacity in comparison to the oleoresin. The uptake of the nanoparticles was studied in cell model systems through confocal laser microscopy and flow cytometry that indicated a probable energy-dependent mechanism

Allergens from Edible Insects: Cross-reactivity and Effects of Processing

Purpose of Review The recent introduction of edible insects in Western countries has raised concerns about their safety in terms of allergenic reactions. The characterization of insect allergens, the sensitization and cross-reactivity mechanisms, and the effects of food processing represent crucial information for risk assessment. Recent Findings Allergic reactions to different insects and cross-reactivity with crustacean and inhalant allergens have been described, with the identification of new IgE-binding proteins besides well-known pan-allergens. Depending on the route of sensitization, different potential allergens seem to be involved. Food processing may affect the solubility and the immunoreactivity of insect allergens, with results depending on species and type of proteins. Chemical/enzymatic hydrolysis, in some cases, abolishes immunoreactivity. Summary More studies based on subjects with a confirmed insect allergy are necessary to identify major and minor allergens and the role of the route of sensitization. The effects of processing need to be further investigated to assess the risk associated with the ingestion of insect-containing food products

Phagocytosis of Astaxanthin-Loaded Microparticles Modulates TGFβ Production and Intracellular ROS Levels in J774A.1 Macrophages

Radiation-induced fibrosis is a serious long-lasting side effect of radiation therapy. Central to this condition is the role of macrophages that, activated by radiation-induced reactive oxygen species and tissue cell damage, produce pro-inflammatory cytokines, such as transforming growth factor beta (TGFb). This, in turn, recruits fibroblasts at the site of the lesion that initiates fibrosis. We investigated whether astaxanthin, an antioxidant molecule extracted from marine and freshwater organisms, could help control macrophage activation. To this purpose, we encapsulated food-grade astaxanthin from Haematococcus pluvialis into micrometer-sized whey protein particles to specifically target macrophages that can uptake material within this size range by phagocytosis. The data show that astaxanthin-loaded microparticles are resistant to radiation, are well-tolerated by J774A.1 macrophages, induce in these cells a significant reduction of intracellular reactive oxygen species and inhibit the release of active TGFb as evaluated in a bioassay with transformed MFB-F11 fibroblasts. Micro-encapsulation of bioactive molecules is a promising strategy to specifically target phagocytic cells and modulate their own functions

Effects of Combination Treatments with Astaxanthin-Loaded Microparticles and Pentoxifylline on Intracellular ROS and Radiosensitivity of J774A.1 Macrophages

Radiation-induced fibrosis (RIF) is a serious, yet incurable, complication of external beam radiation therapy for the treatment of cancer. Macrophages are key cellular actors in RIF because of their ability to produce reactive oxidants, such as reactive oxygen species (ROS) and inflammatory cytokines that, in turn, are the drivers of pro-fibrotic pathways. In a previous work, we showed that phagocytosis could be exploited to deliver the potent natural antioxidant astaxanthin specifically to macrophages. For this purpose, astaxanthin encapsulated into \u3bcm-sized protein particles could spe- cifically target macrophages that can uptake the particles by phagocytosis. In these cells, astaxanthin microparticles significantly reduced intracellular ROS levels and the secretion of bioactive TGF\u3b2 and increased cell survival after radiation treatments. Here we show that pentoxifylline, a drug currently used for the treatment of muscle pain resulting from peripheral artery disease, amplifies the effects of astaxanthin microparticles on J774A.1 macrophages. Combination treatments with pentoxifylline and encapsulated astaxanthin might reduce the risk of RIF in cancer patients

Lipid exchange in mitochondrial cytochrome c release: pro-apoptotic effect of maize lipid transfer protein

Membrane lipids and protein-lipid interactions are attracting increasing interest in the field of cell death and apoptosis. Some pro-apoptotic proteins, like Bid, appear to have an intrinsic capacity of binding and exchange lipids but it is still unclear whether this function could be relevant for apoptotic signalling cascade. We have studied the ability of a plant lipid transfer protein, not related to animal apoptotic cascade, to induce cytochrome c release from mammalian mitochondria. Non -specific lipid transfer proteins (nsLTPs) are ubiquitous plant proteins that have been shown to bind, in vitro, various amphiphilic molecules including lysolipids and glycolipids and to facilitate in vitro transfer of phospholipids between membranes. The results showed that, in the presence of specific lipid molecules (i.e. lysolipids), ns-LTP from maize is able to induce cytochrome c release from the intermembrane space of mouse liver mitochondria. These data are discussed with respect to the role played by lipids and lipid binding in apoptosis

Turning a green alga red: engineering astaxanthin biosynthesis by intragenic pseudogene revival in Chlamydomonas reinhardtii.

SummaryThe green alga Chlamydomonas reinhardtii does not synthesize high-value ketocarotenoids like canthaxanthin and astaxanthin, however, a β-carotene ketolase (CrBKT) can be found in its genome. CrBKT is poorly expressed, contains a long C-terminal extension not found in homologues and likely represents a pseudogene in this alga. Here, we used synthetic re-design of this gene to enable its constitutive overexpression from the nuclear genome of C. reinhardtii. Overexpression of the optimized CrBKT extended native carotenoid biosynthesis to generate ketocarotenoids in the algal host causing noticeable changes the green algal colour to a reddish-brown. We found that up to 50% of native carotenoids could be converted into astaxanthin and more than 70% into other ketocarotenoids by robust CrBKT overexpression. Modification of the carotenoid metabolism did not impair growth or biomass productivity of C. reinhardtii, even at high light intensities. Under different growth conditions, the best performing CrBKT overexpression strain was found to reach ketocarotenoid productivities up to 4.5 mg L-1 day-1. Astaxanthin productivity in engineered C. reinhardtii shown here is competitive with that reported for Haematococcus lacustris (formerly pluvialis) which is currently the main organism cultivated for industrial astaxanthin production. In addition, the extractability and bio-accessibility of these pigments was much higher in cell wall deficient C. reinhardtii than the resting cysts of H. lacustris. Engineered C. reinhardtii strains could thus be a promising alternative to natural astaxanthin producing algal strains and may open the possibility of other tailor-made pigments from this host

Influence of Tilia tomentosa Moench Extract on Mouse Small Intestine Neuromuscular Contractility

Functional gastrointestinal disorders (FGIDs) are characterized by abdominal pain, bloating and bowel disturbances. FGID therapy is primarily symptomatic, including treatment with herbal remedies. Flower extract of Tilia tomentosa Moench (TtM) is occasionally used as an anti-spasmodic in popular medicine. Since its effect on intestinal response is unknown, we evaluated the influence of TtM extract on small intestine contractility. Ileal preparations from C57BL/6J mice were mounted in organ baths to assess changes in muscle tension, following addition of TtM extract (0.5-36 mu g/mL) or a vehicle (ethanol). Changes in contractile response to receptor- and non-receptor-mediated stimuli were assessed in ileal preparations pretreated with 12 mu g/mL TtM. Alterations in the enteric nervous system neuroglial network were analyzed by confocal immunofluorescence. Increasing addition of TtM induced a marked relaxation in ileal specimens compared to the vehicle. Pretreatment with TtM affected cholinergic and tachykininergic neuromuscular contractions as well as K+-induced smooth muscle depolarization. Following incubation with TtM, a significant reduction in non-adrenergic non-cholinergic-mediated relaxation sensitive to N omega-Nitro-L-arginine methyl ester hydrochloride (pan-nitric oxide synthase inhibitor) was found. In vitro incubation of intestinal specimens with TtM did not affect the myenteric plexus neuroglial network. Our findings show that TtM-induced intestinal relaxation is mediated by nitric oxide pathways, providing a pharmacological basis for the use of TtM in FGIDs

Spray-drying Microencapsulation of an Extract from Tilia tomentosa Moench Flowers: Physicochemical Characterization and in Vitro Intestinal Activity

Silver linden (Tilia tomentosa Moench, TtM) flowers possess several health-promoting properties, especially at the neurological level, such as intestinal relaxation activity associated with specific flavonols, particularly quercetin and kaempferol derivatives. However, such molecules are susceptible to degradation upon different triggers like heat, light and extreme pH values. To overcome the scarce stability of TtM flowers bioactive molecules and make them suitable for developing functional food and supplements, we applied microencapsulation. Spray-drying microencapsulation of TtM flowers extract was performed using three starch-derived wall materials: maltodextrin 12 DE (MD12) and 19 DE (MD19), and OSA-modified starch (OSA-S). The stability of total phenols, flavanols, and antioxidant capacity was monitored for 70 days under accelerated stress conditions (40 °C/70% RH) by HPLC and spectrophotometric methods, and the intestinal contractile activity was tested in a murine model. In comparison to MD12 and MD19, OSA-S stood out for the higher encapsulation efficiency of quercetin and kaempferol glycosides (+ 36-47% compared to MD12 and + 18-24% compared to MD19) and stability thereof (half-life on average + 30% compared to MD12 and + 51% compared to MD19). The intestinal contractile activity of OAS-S powders resulted comparable to the original extract, indicating that flavonols were biologically active and accessible. Our results underly the potential advantages of OSA-S encapsulated formulation as a functional ingredient for the development of nutraceutical products

Allergy to pomegranate

Food allergy is an immunological disease whose prevalence is increasing worldwide. Pomegranate could determine allergic reactions, even if a limited number of cases is reported in literature. In the present chapter, after an introductive part describing the pathogenesis and the clinical aspects of allergy and more specifically food allergy, we present a review of the scientific literature dealing with allergy to pomegranate, examining more in depth the involvement of non specific Lipid Transfer Proteins (nsLTP). Indeed, these proteins represent the only pomegranate allergens characterized at the molecular level

Rheology of individual chitosan and polyphenol/chitosan microparticles for food engineering

Encapsulation of perishable bioactive molecules has become a standard process in the food industry. Beads in the millimeter size range may offer technological advantage over smaller particles and nano-structured materials for encapsulation purposes because of higher volume to surface ratios, but their size might seriously affect the sensory properties of the final edible products. We investigated the rheological properties of individual beads composed of chitosan (CTS) cross-linked with sodium tripolyphosphate (TTP). We produced the beads by the external gelation method using different combinations of CTS and TTP concentrations. Analysis of raw data showed that CTS and TTP affected both the morphology and the response of the particles to mechanical load in a non proportional way. The data indicated that the mechanical properties of the beads could not be explained with the aid of standard linear viscoelastic models. We therefore exploited a recently-developed nonlinear model to analyse mechanical data collected during the stress phase and estimate viscoelastic parameters. We found a clear synergic effect of CTS and TTP concentrations on particles’ stiffness and a crossover effect on their viscosity. Two representative bioactive food phenolic compounds, i.e. chlorogenic acid and catechin, were then encap- sulated into the beads. Encapsulation of chlorogenic acid and catechin dramatically altered the viscoelastic behaviour of the particles in a similar way. The particles became less stiff and viscous and more fluid-like compared to empty beads with the same CTS and TTP composition. Our results pave the way for a more ac- curate evaluation of sensory characteristics of novel functional foods incorporating particle carriers for bioactive molecules